Manuscript received: 07.12.2010 Accepted: 14.05.2011

Turk J Gastroenterol 2012; 23 (4): 333-338doi: 10.4318/tjg.2012.0343

Address for correspondence: Cem KARAGÖZLÜEge University, Faculty of Agriculture Department of Dairy Technology 35100 Bornova, ‹zmir, TurkeyPhone: + 90 232 311 29 02 • Fax: + 90 232 388 18 64E-mail: cem.karagozlu@ege.edu.tr

A study on the protective activity of kefir againstgastric ulcer

Yahya T. ORHAN1, Cem KARAGÖZLÜ2, Sülen SARIO⁄LU1, Osman YILMAZ3, Nergiz MURAT4, Sedef G‹DENER4

Departments of 1Pathology, 3Physiology and 4Pharmacology, Dokuz Eylül University, School of Medicine, ‹zmirDepartment of 2Dairy Technology, Ege University, Faculty of Agriculture, ‹zmir

Amaç: Kefirin nonsteroid anti-inflamatuvar ilaç ile peptik ülser hastal›¤› üzerine, kefirin mide mukus bariyerinin miktar› ve et-kinli¤i üzerindeki etkisi deneysel bir modelle araflt›r›lm›flt›r. Gereç ve Yöntem: 28 Wistar türü erkek s›çan üzerinde yap›lan arafl-t›rmada 14 denek kontrol olarak 15 gün boyunca standart olarak grup bafl›na 300 gram standart s›çan yemi ile beslenmifl, di¤er14 denek ise 7 gün yem al›p bu günden sonra 300 gram yem ile 450 gram kefir kar›flt›r›larak beslenmifltir. Her iki gruptan 7 dene-¤e subkutan 30 mg/kg indometazin verilip 4 saat sonra sakrifiye edilmifltir. Histopatolojik inceleme ile gastrik ülserasyon geliflimiskorlanm›fl ve imaj analizi ile mukus miktar› kesitlerde saptanm›flt›r. Bulgular: Ülserasyon skorlar› karfl›laflt›r›ld›¤›nda kefiralan ve almayan gruplar aras›nda fark yoktur. Mikroskopik incelemede sadece indometazin verilen gruplarda gastrik ülserasyonve erozyon saptanm›flt›r. Kefir almayan kontrol grubunda 4 (%57) olguda erozyon ve 3 (%43) olguda ülserasyon saptanm›flt›r. Ke-fir alan grupta erozyon 6 olguda (%86), ülserasyon 1 (%14) olguda saptanm›flt›r. Gruplar aras›nda istatistiksel fark saptanmam›fl-t›r (Mann Whitney Test p=0.25 ). Kontrol grubunu oluflturan deneklerde kefir al›m› olmaks›z›n, gastrik mukozada mukus salg›s›-n›n bulundu¤u alan yüzdesi % 6.04±2.94 olarak bulunmufltur. Kefir alanlarda ise %5.90±3.15’dir. Kefir almayan ve indometazinalanlarda bu oran %4.65±1.55 olup, kefir alan ve indometazin alanlarda ise %3.75±2.5 bulunmufltur. Bu da istatistiksel analizgruplar aras›nda fark bulunmad›¤›n› ortaya koymufltur (Kruskal Wallis p= 0.313, r=0.52). Sonuç: Bu bulgular kefirin gastrik mu-kus miktar›n› ya da koruyuculu¤u artt›rd›¤›n› desteklememektedir. ‹statistiksel sonuçlar önemsiz ç›ksa da, nonsteroid anti-infla-matuvar ilaç ile ülser yap›lan ve kefir ile beslenen grupta sonuçlar›n daha iyi ç›kmas› bu çal›flman›n daha kapsaml› olarak yeni-den yap›lmas› düflünülebilir.

Anahtar kelimeler: Kefir, probiyotik, gastrik ülser, imaj analiz

Background/aims: The effect of kefir on peptic ulcer disease was evaluated in an experimental model, with non-steroid anti-inf-lammatory drugs, together with the determination of gastric mucus secretion by quantitative digital histochemistry. Materials andMethods: The experimental group included 28 male albino Wistar rats. After a diet with standard rat bait for 7 days, 14 rats we-re fed with kefir for 7 days while the others were kept on the same diet. At the 14th day, indomethacin was injected to 7 of the ratsfed on kefir and to 7 of the rats on standard rat bait. All the rats were sacrificed after 4 hours. Gastric erosion and ulceration werescored histopathologically. Mucosal mucus was quantified by image analysis, and periodic acid-Schiff stained area percentage wasdetermined. Results: Erosion and ulceration were identified only in cases that received indomethacin. In the cases on kefir, erosi-on was identified in 6 cases (86%) and ulceration in 1 case. Rats fed on standard diet had erosion in 4 cases (57%) and ulcerationin 3 (43%), but the difference was statistically insignificant (Mann-Whitney test, p=0.25). The stained area percentage for gastricmucus was not different between the four groups (Kruskal-Wallis test, p=0.313). Conclusions: These findings suggest that kefir do-es not change gastric mucus secretion. Although statistically insignificant, as there were more cases with ulceration in cases on therat diet, kefir might have a beneficial effect on peptic ulcer disease induced by non-steroid anti-inflammatory drug. This requiresfurther evaluation in larger series.

Key words: Kefir, probiotics, gastric ulcer, image analysis

ORIGINAL ARTICLE

Gastirik ülsere karfl› kefirin koruyucu aktivitesi üzerine bir araflt›rma

INTRODUCTION

Gastric ulceration is related to more than one fac-tor, including Helicobacter pylori (H. pylori) infec-tion, stress, mucosal mucus secretion, gastric irri-tants, and gastric acidity. It has been claimed formany years that the consumption of kefir, a probio-tic fermented milk product, is beneficial for the tre-atment of gastric and duodenal ulcer. Upon thisobservation, Evenshtein (1) described in 1978 thatthe gastric juice was increased by 89% after con-sumption of kefir for six weeks. It was also de-monstrated that kefir reduced H. pylori infectionowing to its antibacterial effects in vivo (2,3). Onthe other hand, to the best of our knowledge, theeffect of kefir on the gastric mucosal mucus secre-tion, which may be a factor inducing ulceration,has not been described yet. It is thought that an in-crease in gastric mucosal secretion might delay orprevent the disease (4). In the present work, it wasintended to evaluate the effect of kefir consumpti-on in an experimental model of acute gastric ulcer,with non-steroid anti-inflammatory drugs (NSA-IDs), with determination of gastric mucus secreti-on by quantitative digital histochemistry.

MATERIALS AND METHODS

Experimental Materials and Animals

The study protocol was approved by the Local Et-hics Committee at Dokuz Eylül University Fa-culty of Medicine. The experimental groups con-sisted of 28 male albino Wistar rats from DokuzEylül University, Medical Science Faculty, Labo-ratory of the Experimental Animals Department.After a diet with standard rat bait for 7 days, 14rats were fed with kefir for 7 days while the otherswere kept on the same diet. At the 14th day of theexperiment, 7 rats fed on kefir and 7 rats fed stan-dard rat bait were given indomethacin injection,and after 4 hours (h) all the rats were sacrificed(Table 1).

The rats were weighed on the 1st, 7th and 14th days(5,6).

Production of Kefir

Raw cow’s milk supplied from Ege University Ag-riculture Faculty, Menemen Research and Appli-cation Farm, was used in kefir-making. The milkwas heated at 90°C for 5 minutes (min), followedby inoculation with the kefir grains provided byEge University Agriculture Faculty, Dairy Tech-nology Department, Microbiology Laboratory, at arate of 5% and incubated at 20±3°C until pH 4.7was attained in glass cups. After incubation, kefirgrains were removed by a sterile wire sieve. Thefiltrate was used as kefir in the rest of the experi-ment (7).

Raw Milk and Kefir Analysis

In raw milk and kefir, the pH, titratable acidity(expressed as percentage lactic acid), total solids,fat, total nitrogen, ash, and lactose contents weredetermined according to Turkish Standards Insti-tution Method (8). In the counts of Streptococcusspp., M17 agar (OXOID, CM 785), Lactobacillusspp. MRS agar (OXOID, CM 361) and total yeastsDichloran Rose Bengal Chloramphenicol (DRBC)agar (CM 727) were used (9).

Gross Examination

The gastric and duodenal tissues were evaluatedby gross examination and scored as follows: 0: noevidence of ulceration, 1: erosion, and 2: ulcerati-on. The gastric and duodenal tissues were forma-lin-fixed, and for histopathological examination,the gastric wall was sampled, beginning from thecardia-esophageal junction to the duodenum thro-ugh the lesser curvature, and paraffin-embeddedtissue blocks were prepared. The sections fromthese blocks were stained by hematoxylin and eo-sin (H&E) and periodic acid-Schiff (PAS) (10).

Microscopic Evaluation of Gastric Mucosa

Hematoxylin and eosin-stained sections were eva-luated for any evidence of erosion or ulcerationand scored as follows: 0: no evidence of erosion orulceration, 1: erosion identified by loss of the su-perficial layer of mucosa replaced by inflamma-

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Group 1 Group 2 Group 3 Group 4Nutrition Kefir + standard bait + Standard bait + Kefir + standard bait Standard bait +

tap water tap water tap water tap water

Formation of ulcer Indomethacin Indomethacin - -

Duration to sacrifice 14 days + 14 days + 14 days + 14 days +4 hours 4 hours

Table 1. The distribution of the experimental rats

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FFiigguurree 11.. aa.. The gastric mucosa with superficial erosion. bb.. Gas-tric ulcer, with total loss of the mucosa (H&E, original magnifi-cation x10).

tory cells and/or fibrin, and 2: ulceration with lossof mucosal layer. Additionally, H. pylori was se-arched in every section (10,11,12). In the sectionsof the whole gastric mucosa, painted hematoksileneosin was processed and researched. In thoseparts there were not gastric erosion and ulcerati-on, but erosion and ulceration was scored to comeinto (Fig. 1a - b, 2).

Determination of Gastric Mucus by Quantitative Digital Histochemistry

For a standard measurement of the gastric muco-sal mucus, PAS-stained sections from the smallcurvature were selected for measurement, andimages were taken subsequently from the gastro-esophageal junction to the duodenum. All the gas-tric mucosal mucus could thus be quantified at thelesser curvature, excluding the zones with extensi-ve erosion and ulceration, where mucosa could notbe observed. Digital images were obtained fromthe selected areas using light microscopy (Oly-mpus, BX51, Japan) at original magnification ofx40. Images were captured by a digital color vide-o camera (Olympus DP70). The stained mucosalareas in the captured images were quantified bymeasuring the high power fields using a software(Mustafa Sakar, ‹zmir, Turkey). The magenta co-lor was selected; the image analysis program cal-culated the stained area percentage (SAP), as des-cribed previously (11-13). On the image when themagenta color was selected, neighbors to that anddesignated color neighbors were covered by theanalysis programme automatically, and was ex-pressed as the proportion of the selected area tothe total area (Fig. 3 a - b) (12).

Statistical Evaluation

Statistical evaluation was performed using theStatistical Package for the Social Sciences for

Windows software package (SPSS ver. 11.0 forWindows; Chicago, IL). SPSS version 11.0 wasused for statistical analysis and the Mann–Whit-ney U test for comparisons between groups. Therelationship between variables was investigatedusing Pearson’s rank correlation (14).

RESULTS

The chemical and microbiological properties ofraw milk and kefir are summarized in Table 2.

The average weight of the rats was 188.04±19.66g, 210.46±21.20 g and 219.39±22.59 g on the 1st, 7th

and 14th day, respectively, and there was a signifi-cant difference between groups (p<0.000, one wayANOVA). There was a significant difference thefirst and second as well as between the first andthird measurements (p<<0.000 and p<<0.000,Mann-Whitney U test), but there was no signifi-cant difference between the second and third me-asurements (p=0.14, Mann-Whitney U test).

Raw milk KefirpH 6.7±0.04 4.5±0.06

Total solids (%) 11.30±0.8 10.78±0.85

Acidity (lactic acid), % 0.13±0.03 0.79±0.15

Milk fat (%) 3.2±0.2 3.15±0.35

Lactose (%) 3.8±0.3 3.45±0.20

Protein (%) 3.5±0.2 3.40±0.15

Ash (%) 0.80±0.05 0.75±0.04

Lactococcus spp. (cfu/ml) - 2.25x108

Streptococcus spp. (cfu/ml) - 1.5x108

Yeast (cfu/ml) - 1.3x106

Table 2. The chemical and microbiological properti-es of raw milk and kefir samples (n=7)

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FFiigguurree 33 aa--bb.. The demonstration of the stained gastric mucusand the image analysis for measuring the stained area percentage(PAS x10).

FFiigguurree 22.. Ulceration damaging the whole mucosa and destro-ying the superficial muscle layer (H&E original magnificationx4).

When the body masses of groups on standard dietand those receiving kefir were compared, no signi-ficant difference was noted in the last two measu-rements (p=0.10 and p=0.19, respectively). Grossand microscopic examinations did not reveal erosi-on or ulceration for the cases that did not receiveindomethacin. There was erosion in 4 (57%) andulceration in 3 (43%) of the cases fed on standarddiet, while erosion was identified in 6 (86%) andulceration in 1 (14%) of the cases receiving kefir(Table 3). There was no significant difference bet-ween the groups (p=0.25, Mann-Whitney U test).

The average SAP for all cases was 5.09±2.64%(range: 0.99-12-10%). The mean SAP for cases re-ceiving standard diet and kefir diet, but not indo-methacin, was 6.40±2.94 and 5.90±3.15, respecti-vely, and for the groups receiving indomethacin,these values were 4.65±1.55 and 3.75±2.5 (Table3). There was no significant difference betweenthe groups (p=0.313, Kruskal-Wallis test). Ove-rall, when the 14 cases on standard diet were com-pared with the cases on kefir diet, there was nosignificant difference between the groups in termsof SAP values (p=0.32).

DISCUSSION

Peptic ulcer disease is a result of the loss of balan-ce between gastric mucosal protection and gastricacidity. The mucus layer spares the gastric muco-sal surface and pit epithelium from the digestiveeffect of the gastric juice.

The leading etiologic factor for the development ofpeptic ulcer disease, as well as of gastric carcino-ma and mucosa-associated lymphoid tissue(MALT) lymphoma is H. pylori infection. The bac-teria act on the 1500 proteins, which induces epit-helial cell apoptosis, metaplasia, atrophy, dyspla-sia, and carcinoma, as well as ulceration followingmultiple pathways. The latter is located at the du-odenum predominantly, in the setting of antral H.pylori infection, with increased gastric acidity andgastric metaplasia of the duodenal mucosa (15).Many drugs as well as natural products have beenevaluated for the eradication of H. pylori since theidentification by Marshall and Warren and Lich-tenberger et al. (16,24).

Researches on fermented milk and probiotics haveproven the health benefits of these products. Kefiris a probiotic fermented milk product originatingfrom the Caucasian mountains. It is a culturedmilk, in which both acid and alcohol fermentationis developed. Kefir has a refreshing and slightly

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Table 3. The mean values of body mass measurements and histopathological evaluation results of the four ex-perimental groups of rats

Standard diet Standard diet Kefir diet Kefir dietindomethacin indomethacin indomethacin indomethacin

- + - +Body mass 1 (gram) 202.14 198.57 184.28 168.57

Body mass 2 (gram) 211.28 223.0 207.14 201.85

Body mass 3 (gram) 219.42 224.57 217.14 216.42

Erosion none 4 (57%) none 6 (86%)

Ulceration none 3 (43%) none 1 (14%)

Stained area percentage (%) 6.40±2.94 4.65±1.55 5.90±3.15 3.75±2.5

sharp taste and aroma. The original starter of ke-fir is also called kefir grain. Kefir grain, resemb-ling florets of a cauliflower, are white-yellowish incolor, 2 to 4 mm in diameter. The surface of thegrain is rough and convoluted. They are held to-gether in clusters as an immobilized system of acomplex community of yeast and bacterial cellsembedded in a microbe-produced polysaccharidematrix along with some milk fat and denaturedmilk protein (7,17). Kefir grain mainly consists oflactose-fermenting bacteria Lactobacillus and Le-uconostoc species (Lactobacillus casei, L. brevis, L.acidophilus, L. kefir, Leuconostoc mesenteroides,and Leu. mesenteroides subsp. dextranicum) aswell as Lactococcus lactis, Lactococcus lactissubsp. cremoris and other lactic acid bacteria. Theother characteristic organisms are lactose-fermen-ting or non-lactose-fermenting yeasts, mainly inc-luding Candida kefir, Kluyveromyces marxianussubsp. marxianus, Torulaspora delbrueckii, andSaccaromyces cerevisiae. The main products pro-duced by kefir grains are lactic acid, carbon dioxi-de, alcohol, diacetyl and acetaldehyde as well asprotein degradation products. It is known that ke-fir was used in cancer treatment in Russia and isstill being used in patients with gastric problems(3,18-20).

Kefir and other fermented probiotics milk pro-ducts may have an antibacterial effect on H. pylo-ri infection (3,21). On the other hand, there is nosatisfactory data about the effect of kefir on othermechanisms, which may contribute to the progres-sion of the available milieu for the development ofpeptic ulcer. The second most frequent factor forthe development of peptic ulcer disease is NSA-IDs. They induce gastric injury by intracellular ac-cumulation and inhibition of prostaglandinsynthesis (22,23). NSAIDs also reduce the hydrop-hobicity of the mucus gel layer by an insult to the

surface-active phospholipids. It has been shownthat this can be prevented by pre-associating aNSAID with zwitterionic phospholipids (24).

The present study is based on the hypothesis thatkefir may have other effects on the gastric milieuin addition to its antibacterial properties. The ex-perimental model depends upon consumption ofkefir with suitable properties. In Table 2, theanalysis results of the kefir, which were only for 1-2 days, were in agreement with the findings of ot-her researchers regarding the general compositionand microbiologic appearance of kefir; especiallyfrom the perspective of probiotic effect, Lactobacil-lus and Streptococcus genus showed that they we-re of sufficient degree considering the number(5,25-27). The gross composition of raw milk usedin the production of kefir was close to the averagemilk composition (1,3,7,17,26,27).

The experimental model questions the effect of ke-fir consumption on the prevention of NSAID-indu-ced peptic ulcer, and if positive results were obtai-ned, this would lead to a series of new questionsabout the mechanism. We did not find a statisti-cally significant difference between groups on ratbait and kefir considering lesions of erosion andulceration, but the number of cases with ulcerati-on was more for cases that did not receive kefir.This finding may deserve evaluation in a largernumber of cases, as the statistically insignificantresults may have been caused by the low numberof cases in this series.

The increase in the amount of mucus secretionmay reduce the NSAIDs ulcerogenic effect on thegastric mucosa (28). The amount of gastric mucuswas quantified by image analysis based on PAShistochemistry. There was no significant differen-ce between the group on the kefir diet and the gro-up on the standard rat bait (p>0.05).

The results of the present study argue against therole of kefir consumption on prevention of NSAID-induced gastric ulceration by increasing gastricmucus. As the number of cases with ulcerationwas less in cases on kefir in this series, the effects

of kefir on indomethacin-induced gastric injurymight be evaluated in larger series with the othermucosa-saving effects of kefir, like prostaglandinE2 secretion, which is suggested as a mechanismfor decreasing alcohol-induced injury (29).

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